Project Summary In this proposal we aim to study the integrated metabolism of reactive oxygen species (ROS) and energetics, experimentally and by computational modeling, applying two recently introduced concepts: Redox- optimized ROS balance (R-OR balance), and control by diffuse loops. In order to analyze in an integrated manner the mechanisms of control and regulation of energy and ROS balance in an important disease for public health, we will investigate working cardiac muscle in a type 2 diabetes mellitus (T2DM) rat model, focusing on the effects of insulin and metformin upon energy and ROS pathways. In the diabetic cardiac muscle, we seek to understand the interdependence of energy and ROS fluxes and their relation to the redox environment, We will focus on the effects of insulin and metformin (a widely-used anti-hyperglycemic drug) on metabolic control. These studies will apply state of the art quantitative tools of metabolic control analysis based on the inhibitor titration method, and on the analysis of transients after perturbation of the steady state regime. We plan to monitor metabolic variables and ROS in rat cardiac trabeculae loaded with fluorescent indicators, under working conditions in a force transducer device. The experimental results will be used to constrain and fine-tune a computational model of the cardiac myocyte that integrates mechanical, electrophysiological and metabolic activities (ECME model). So far, the ECME model has been able to successfully simulate the behavior of i) oscillations in mitochondrial membrane potential, NADH, glutathione, and ROS, ii) the dynamics of mitochondrial NADH, calcium, and ADP during changes in supply and demand in the heart, and iii) the dynamics of the sarcolemmal membrane potential during mitochondrial oscillations in whole hearts undergoing arrhythmias. The model will be extended to incorporate pathways upstream Acetyl CoA, namely glycolysis, pentose phosphate pathways and beta-oxidation. A more detailed mathematical description of the electron-transport complexes of the respiratory chain, and of the ROS scavenging pathways, will enable accounting for the mechanisms of ROS balance. The computational model will be subjected to metabolic control in an effort to identify the steps that participate in the control and regulation of the network of energy and ROS pathways. We are convinced that in order to perform a rational intervention in the treatment and prevention of a disease regarding the cardiovascular system, a deeper understanding of the integrated behavior of metabolic networks is needed. This justifies our attempt to build a computational model that will lead to a quantitative understanding of the dysfunctional aspects of heart physiology, and point out potential targets that could be used for therapeutic interventions, either pharmacological, nutritional or by gene therapy.